Agent for reduction of scar formation by using wound alkalinization

ABSTRACT

The present invention relates to an agent for reducing scar formation, which inhibits the scar formation by injecting sodium bicarbonate to the dermis tissue of the wound with a syringe to directly control pH of the wound site, namely, by alkalinizing the wound to inactivate TGF-β. According to the present invention, the agent for reducing scar formation by controlling the wound healing process can be clinically and immediately applied to the treatment of tylosis scar and keloid, etc., and is effective to the treatment of intractable fibrosis diseases. Also, the invention can be widely applied in many ways as an easy tool which can control the action of TGF-β in a living organism.

TECHNICAL FIELD

[0001] The present invention relates to an agent for reducing scarformation by using alkalinization of the wound, which is clinically easyto apply. More particularly, the present invention relates to an agentfor reducing scar formation, which inhibits the scar formation byinjecting sodium bicarbonate to the dermis tissue of the wound with asyringe to directly control pH of the wound site, namely, byalkalinizing the wound to inactivate TGF-β.

BACKGROUND ART

[0002] There have been efforts to reduce the formation of scar. However,an effective method has not yet been found. It was reported that thewound of a fetus is cured without scar. After such report, trials havebeen made to reveal the differences between the wound healing process ina fetus and that in a mature individual, change the wound healingprocess in a mature individual to that in a fetus, and thereby reducethe scar formation. One of the revealed differences is that TransformingGrowth Factor-Beta (TGF-β) is not immunochemically detected in the woundof a fetus, but functions in the wound healing process of a matureindividual and plays an important role in the scar formation. This factshows that it would be possible to inhibit the scar formation bycontrolling TGF-β. Actually, there was a report that the scar formationin a white rat could be reduced by the use of an antibody against TGF-β(Acidic cellular environments; activation of latent TGF-beta andsensitization of cellular responses to TGF-beta and EGF. InternationalJournal of Cancer. 43(5):886-91, May 15, 1989). TGF-β is secreted ininactivated state from cells and is mostly present in inactivated stateeven outside of cells. Only when exposed to a certain specificcondition, TGF-β is activated, combines with a receptor of a target celland functions. Heat, acidic condition, protease, etc. are known asphysicochemical conditions which can activate TGF-β (Physicochemicalactivation of recombinant latent transforming growth factor-beta's 1, 2,and 3. Growth Factors. 3(1):35-43, 1990). However, no studies have beenmade yet with respect to the reduction of scar formation by using theinactivation of TGF-β. The present invention aims to control the pH ofthe wound, which is clinically easy to apply, namely, to alkalinize thewound and lead the inactivation of TGF-β, and to confirm whether theinactivation actually reduces the scar formation.

[0003] There have been several trials to reduce scar formation bycontrolling TGF-β which functions during the entire process of scarformation. However, those trials were related to the use of an antibodyand have problems in actual application. TGF-β widely functions in aliving organism and is under active study in various fields. Theprocesses of formation, secretion and function of TGF-β have not yetbeen clearly known. According to the facts discovered to date, TGF-β issecreted from cells in inactivated state and functions with a targetorgan under specific conditions. Heat, acidic pH, and protease are knownas the physicochemical conditions which can activate TGF-β. However, notrials have been made to reduce the scar using TGF-β.

DISCLOSURE OF INVENTION

[0004] The present invention was conceived based upon the above satedfacts. The inventors of the present invention have completed the presentinvention by controlling the pH of the wound, which is clinically easyto apply, that is, by alkalinizing the wound and inactivating TGF-β,thereby leading the wound healing process which is similar to that of afetus and confirming that the scar formation is reduced. Accordingly,the objective of the present invention is to provide an agent forreducing scar formation, which inactivates TGF-β, reduces the scarformation and thus heals the wound without scar.

[0005] The present invention is directed to a method for reducing scarformation during the wound healing process. TGF-β is known to play animportant role in the scar formation process of the wound. To inactivateTGF-β, the wound should be alkalinized. To easily alkalinize the wound,sodium bicarbonate in the concentration of sodium bicarbonate 1 mEq (1cc)/distilled water 10 cc is injected to the dermis tissue of the woundto directly control pH of the wound, thereby reducing the scar formationof the wound. The pH range which effectively reduces the scar formation,namely, which does not activate TGF-β and alkalinizes the wound, is9.0-10.0, preferably, 9.2-9.8. Also, sodium bicarbonate of the presentinvention reduces fibrosis in the process of tissue regeneration orhealing, by controlling pH (alkalinization) of the regeneration orhealing spot.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006]FIG. 1a shows the tissue of the control group after two weeks fromthe injection of distilled water, instead of sodium bicarbonate(dyeing:hematoxylin-erosin dyeing, magnification: 40×).

[0007]FIG. 1b shows the tissue of the test group after two weeks fromthe injection of sodium bicarbonate(dyeing: hematoxylin-erosin dyeing,magnification: 40×).

[0008]FIG. 2a shows the tissue of the control group after two weeks fromthe injection of distilled water, instead of sodium bicarbonate(dyeing:trichrome dyeing, magnification: 400×).

[0009]FIG. 2b shows the tissue of the test group after two weeks fromthe injection of sodium bicarbonate(dyeing: trichrome dyeing,magnification: 400×).

[0010]FIG. 3a shows the tissue of the control group after eight weeksfrom the injection of distilled water, instead of sodiumbicarbonate(dyeing: hematoxylin-erosin dyeing, magnification: 40×).

[0011]FIG. 3b shows the tissue of the test group after eight weeks fromthe injection of sodium bicarbonate(dyeing: hematoxylin-erosin dyeing,magnification: 40×).

[0012]FIG. 4a shows the tissue of the control group after eight weeksfrom the injection of distilled water, instead of sodiumbicarbonate(dyeing: trichrome dyeing, magnification: 400×).

[0013]FIG. 4b shows the tissue of the test group after eight weeks fromthe injection of sodium bicarbonate(dyeing: trichrome dyeing,magnification: 400×).

[0014]FIG. 5a shows the tissue of the test group after two weeks fromthe injection of TGF-β (dyeing: hematoxylin-erosin dyeing,magnification: 40×).

[0015]FIG. 5b shows the tissue of the test group after two weeks fromthe injection of both TGF-β and sodium bicarbonate(dyeing:hematoxylin-erosin dyeing, magnification: 40×).

[0016]FIG. 6a shows the tissue of the test group after eight weeks fromthe injection of TGF-β (dyeing: hematoxylin-erosin dyeing,magnification: 100×).

[0017]FIG. 6b shows the tissue of the test group after eight weeks fromthe injection of both TGF-β and sodium bicarbonate(dyeing:hematoxylin-erosin dyeing, magnification: 100×).

[0018]FIG. 7a shows the tissue of the test group after eight weeks fromthe injection of TGF-β (dyeing: trichrome dyeing, magnification: 400×).

[0019]FIG. 7b shows the tissue of the test group after eight weeks fromthe injection of both TGF-β and sodium bicarbonate(dyeing: trichromedyeing, magnification: 400×).

EXAMPLES

[0020] The following examples disclose the present invention in moredetail.

[0021] In the wound healing process of an adult white rat, it washistologically studied whether the alkalinization of the wound reducesthe scar formation, and it was indirectly confirmed whether such areduction is caused by TGF-β inactivation, by observing differences ofscar leading action of TGF-β between the alkalinized wound and thecontrol wound.

[0022] Materials

[0023] Animal: forty white rats of Spraue-Dawley origin, which weigh300-350 mg

[0024] bovine TGF-β

[0025] 0.1 mEq sodium bicarbonate

[0026] Methods of Experiment

[0027] A. Experiment of Wound Alkalinization (Twenty White Rats)

[0028] On the dorsum of adult white rats, skin near the limbs which areon the same distance from the median line was incised in about 10 mmlong. After incision, the control group was simply sutured. In shamcontrol group, 0.1 cc distilled water was injected to the dermis of theincised wound. In test group, 0.1 mEq sodium bicarbonate 0.1 cc (Ph9.5±0.3) was injected to the dermis of the incised wound.

[0029] a. control×1 site: suture only

[0030] b. experimental×1 sites: 0.1 mEq Sodium bicarbonate 0.1 ccinjection and suture

[0031] B. Experiment of TGF-β Inactivation (Twenty White Rats)

[0032] On the dorsum of adult white rats, skin near the limbs which areon the same distance from the median line was incised in about 10 mmlong. Before suture, TGF-β 0.05 μg was injected to the dermis of theincised wound in test group I, while both TGF-β 0.05 μg and 0.1 mEqsodium bicarbonate 0.1 cc (Ph 9.5±0.3) were injected to the dermis ofthe incised wound in test group II.

[0033] a. experimental I×1 site: TGF-β injection and suture

[0034] b. experimental II×1 sites: TGF-β+sodium bicarbonate injectionand suture

[0035] Analysis of the Results

[0036] A. Histological Observation

[0037] On 14^(th) and 56^(th) days from the experiments, tissues of tenadult white rats were taken, fixed in 10% neutral buffer formalin for 24hours, embedded in paraffin throughout the normal tissue treatingprocess. In the middle of the wound, the tissue was cut into 4 μm thick,perpendicularly to the major axis, and dyed with hematoxyline-eosine andMasson's trichrome to observe the connective tissues (FIGS. 1a to 7 b).

[0038] In FIGS. 1a and 1 b, control group (a) shows a thicker scar,which is marked with an arrow, than the test group (b).

[0039] In FIGS. 2a and 2 b, control group (a) shows thin, compact,irregular blue-died collagen fibers, which are typical collagenappearance of scar. On the other hand, test group (b) shows relativelythick, regular collagen fibers, which are relatively spacious comparedto the control group (a).

[0040] In FIGS. 3a and 3 b, control group (a) shows a thicker scar,which is marked with an arrow, than the test group (b).

[0041] In FIGS. 4a and 4 b, control group (a) shows thin, irregularblue-died collagen fibers, which are typical collagen appearance ofscar. On the other hand, test group (b) shows relatively thick, regularcollagen fibers, of which structures are almost similar to the normaldermis nearby.

[0042] In FIGS. 5a and 5 b, TGF-β injected group (a) shows a thickerscar, which is marked with an arrow, than the test group (b).

[0043] In FIGS. 6a and 6 b, TGF-β injected group (a) shows a thickerscar, which is marked with an arrow, than TGF-β+sodium bicarbonateinjected group (b). The scar of TGF-β+sodium bicarbonate injected group(b) is too thin to find, and the structure of the tissue is similar tothe normal dermis nearby.

[0044] In FIGS. 7a and 7 b, TGF-β injected group (a) shows thin,irregular blue-died collagen fibers, which are typical collagenappearance of scar, and forms a thick scar. On the other hand,TGF-β+sodium bicarbonate injected group (b) shows very thin scar and itscollagen fibers are thick, regular and of which structure is almostsimilar to the collagen structure of normal dermis nearby.

[0045] B. Measurment of the Width of Scar

[0046] With a tissue sample, which was cut perpendicularly to the middleof the wound, the width of the scar by use of image analysis program(Image-Pro version 3.0, Microsoft) under 40 magnification was measured,and the differences between the groups were compared. The result of themeasurement was confirmed by Student t-test.

[0047] Results

[0048] The tissues to be tested were obtained from tested forty whiterats, twenty of which were sacrificed at each time, after 2 and 8 weeksfrom the operational handling.

[0049] A. Experiment of Wound Alkalinization

[0050] After two (2) weeks from a wound was made, an inflammation wasobserved from the epidermal tissue layer to the muscular tissue layer atthe low magnification (×40) in hematoxylin-eosin dyeing, and there wasnot much difference between the degree of deposition of inflammatorycells of the test group and the control group. But the width of the scarof test group was thinner than that of the control group (Refer to FIG.1). The deposited Collagen of Trichrome dyeing was thin and irregular inboth the test group and the control group. But the amount of depositionof the Collagen of the test group was meager compared to that of thecontrol group, and deposition of the Collagen of the control group waslarger and denser (Refer to FIG. 2).

[0051] When the width of scar was measured at magnification of 40, thatof the control group was average of 68.44 μm±22.84, and that of the testgroup was average of 26.66 μm±9.60. As a result of student t-test, thevalue of p at a significant level of 0.05 of the two groups was thevalue of 0.01 or less, which was statistically significant difference(Refer to Table 1).

[0052] In the hematoxylin-eosin dyeing of the wound after eight (8)weeks from the wound was made, the test group formed a thin andunnoticeable scar and the control group formed a thick and significantscar at the low magnification (×40) (Refer to FIG. 3). At the highmagnification (×400), the test group showed thicker and regular Collagenfibers which was not much different from the normal dermis nearby,whereas the control group showed compactly deposited Collagen fiber,which was clearly distinguishable from the normal dermis nearby. Notmany of the fibroblast cells were found in the test group but many ofthem were still found in the control group. In Trichrome dyeing, thedensity of Collagen fiber in the test group was not different from thatof the normal dermis nearby. The fiber was thick and regular, which wasrecovered closed to the dermis, while since thin and irregular fiber wasdensely deposited, the control group clearly showed the scar (Refer toFIG. 4). When the width of the scar was measured at 40 magnification,that of the control group was average of 37.67 μm±7.98, and that of thetest group was average of 13.89 μm±2.42. As a result of student t-test,the value of p at a significant level of 0.05 of the two groups was thevalue of 0.01 or less, which was a statistically significant difference(Refer to Table 1). TABLE 1 Comparison of the width of sodiumbicarbonate group and that of control group (measured by using imageanalysis program, “Image-Pro version 3.0, Microsoft”) Control Group*sodium bicarbonate Value of p (n = 10) group* (n = 10) 2 Weeks 68.44 μm± 22.84 26.66 μm ± 9.60 0.01≧ 8 Weeks 37.67 μm ± 7.98 13.89 μm ± 2.42.0.01≧

[0053] B. Experiment of TGF-β Inactivation

[0054] After two (2) weeks from a wound was made, an inflammation wasobserved from epidermal tissue layer to the muscular tissue layer at thelow magnification (×40) in hematoxylin-eosin dyeing, and there was notmuch difference between the degree of deposition of inflammatory cellsof TGF-β group and sodium bicarbonate+TGF-β group. But the width of thescar of sodium bicarbonate+TGF-β group was thinner than that of theTGF-β group (Refer to FIG. 5). The deposited Collagen of Trichromedyeing was thin and irregular in both the test group and the controlgroup. But the amount of deposition of the Collagen of the test groupwas meager compared to that of the control group, and deposition of theCollagen of the control group was larger and denser.

[0055] When the width of scar is measured at magnification of 40, thatof the TGF-β group is average of 74.84 μm±15.93, and that of sodiumbicarbonate+TGF-β group was average of 35.41 μm±6.90. As a result ofstudent t-test, the value of p at a significant level of 0.05 of the twogroups was the value of 0.01 or less, which was a statisticallysignificant difference (Refer to Table 2).

[0056] In the hematoxylin-eosin dyeing of the wound after eight (8)weeks from the wound was made, sodium bicarbonate+TGF-β group formed athinner and less noticeable scar than TGF-β group at the lowmagnification (×40). The control group formed a thicker and moresignificant scar than the test group at the low magnification (×40)(Refer to FIG. 6). At the high magnification (×400) as same as the testof alkalinization of a wound, sodium bicarbonate+TGF-β group showedthicker and regular Collagen fibers which was not much different fromthe normal dermis nearby.

[0057] In Trichrome dyeing, the density of Collagen fiber in sodiumbicarbonate+TGF-β group was not that different from the normal dermistissue nearby. The fiber was thick and regular, which was recoveredclosed to the dermis tissue, while since thin and irregular fiber wasdensely deposited, TGF-β group clearly showed the scar (Refer to FIG.7). When the width of the scar was measured at 40 magnification, that ofTGF-β group was average of 72.92 μm±16.04, and that of sodiumbicarbonate+TGF-β group was average of 49.09 μm±5.58. As a result ofstudent t-test, the value of p at a significant level of 0.05 of the twogroups was the value of 0.01 or less, which was a statisticallysignificant difference (Refer to Table 2). TABLE 2 Comparison of thewidth of sodium bicarbonate group and that of the control group(measured by using image analysis program, “Image-Pro version 3.0,Microsoft”) sodium bicarbonate + TGF-β*(n = 10) TGF-β* (n = 10) Value ofp 2 Weeks 74.84 μm ± 15.93 35.41 μm ± 6.90 0.01≧ 8 Weeks 72.92 μm ±16.04 49.09 μm ± 5.58 0.01≧

INDUSTRIAL APPLICABILITY

[0058] As explained and shown in the Examples above, the agent forreducing scar formation according to the present invention bycontrolling the wound healing process can be clinically and immediatelyapplied to the treatment of tylosis scar and keloid, etc., and iseffective to the treatment of intractable fibrosis diseases. Also, theinvention can be widely applied in many ways as an easy tool which cancontrol the action of TGF-β in a living organism.

1. An agent for reducing scar formation on wound comprising sodiumbicarbonate, characterized by injecting sodium bicarbonate to the dermistissue of the wound to directly control pH of the wound and heal thewound without scar.
 2. The agent for reducing scar formation accordingto claim 1, wherein the reduction of scar formation is achieved bycontrolling pH of the wound to inactivate TGF-β.
 3. The agent forreducing scar formation according to claim 1, wherein the pH of thewound is controlled to range from 9.0 to 10.0.
 4. The agent for reducingscar formation according to claim 3, wherein the pH of the wound iscontrolled to range from 9.2 to 9.8.
 5. The agent for reducing scarformation according to any one of claims 1 to 4, wherein sodiumbicarbonate is used in concentration of 1 mEq (1 cc)/distilled water 10cc.